Collagen IV of basement membrane: V. Bromide-mediated sulfilimine bonds interlock the quaternary structure of NC1-hexamer of scaffolds enabling metazoan evolution.

Collagen-IV (Col-IV) scaffolds, a primordial basement membrane component, enabled animal multicellularity, evolution and adaptation. These scaffolds provide tensile strength and tether macromolecules, forming supramolecular complexes that interact with cell-surface receptors and influence cell-behavior. Triple-helical Col-IV protomers, composed of three α-chains, with a trimeric globular NC1-domain at the C-terminus, oligomerize forming a NC1-hexamer structure that connects adjoining protomers of Col-IV^α121, Col-IV^α556-α121, and Col-IV^α345 scaffolds. Hexamer formation and stability are driven by the extracellular chloride concentration- "chloride pressure". Hexamer structure is reinforced by six sulfilimine bonds forming covalent crosslinks that weld together trimeric NC1-domains of adjoining protomers. We recently found evidence that sulfilimine bonds, independent of chloride, stabilize the quaternary structure of the Col-IV^α345 hexamer of the Col-IV^α345 scaffold. Here, we sought to determine whether this function also pertains to the Col-IV^α121 scaffold that occurs ubiquitously across the animal kingdom, and whether bromine, a cofactor of peroxidasin in bond formation, are evolutionary conserved. We found that sulfilimine bonds stabilized the quaternary structure of the Col-IV^α121 hexamer of bovine, mouse and a basal cnidarian, <i>Nematostella vectensis</i>, and that the mechanism of bond formation mediated by peroxidasin and bromide is evolutionary conserved. Analyses of the crystal structure of the NC1-hexamer revealed that sulfilimine bonds covalently fasten a clasp-motif across the trimer-trimer interface, interlocking the domain-swapping region of neighboring subunits, which reinforces the hexamer quaternary structure imposed by chloride conformational constraints. Collectively, our findings reveal that the sulfilimine-bond reinforcement is a critical event in Col-IV scaffold assembly enabling multicellularity, evolution and adaptation of metazoans, beginning with ancient cnidarians.